Dolastatin 15, a known and potent antineoplastic constituent of the Indian Ocean shell-less mollusk Dolabella auricularia, was utilized as the lead substance from which were developed a series of novel derivatives. The present invention relates to methods of synthetically producing these new agents and presents their in vitro evaluations against a variety of murine and human cancer cell lines, and against a selection of bacteria and fungi. The effect of these derivatives on the inhibition of tubulin polymerization is also disclosed. Surprisingly, all of the new compounds, in which the C-terminal (S)-dolapyrrolidinone unit (Dpy, 5) of Dolastatin 15 is replaced with a series of structurally diverse, more readily available and less expensive amides, show cancer cell growth inhibition activities which are quite comparable to those of Dolastatin 15 (see: U.S. Pat. No. 4,879,278, Pettit et al.) All of the new compounds were, however, less potent than Dolastatin 15 as inhibitors of tubulin polymerization. The structurally modified peptides also caused mitotic arrest in cultured cells and inhibited the growth of a Gram-negative bacterium.
Some of this work was funded by Outstanding Investigator Grant CA-44344-01-08 awarded by the Division of Cancer Treatment, National Cancer Institute, DHHS. The United States government may have certain rights to this invention.
Marine organisms are an exceptionally productive source of biologically active and medicinally important substances bearing unique structures (see: FAULKNER, D. J. 1994, Marine Natural Products, Natural Products Reports, 11, 355; KOBAYASHI, M., et al. 1994, Bioactive substances isolated from marine sponge, a miniature conglomerate of various organisms, Pure and Applied Chemistry, 819; and, Kxc3x96NIG, G. et al. 1994, Biological activities of selected marine natural products, Planta Medica, 60, 532-537). Illustrative are the Indian Ocean (see: PETTIT et al. 1993, The isolation of dolastatins 10-15 from the marine mollusk Dolabella auricularia, Tetrahedron, 49, 9151) and Japanese (see: NAKAMURA et al., 1995, Stereochemistry and total synthesis of Dolastatin E. Tetrahedron Letters, 36, 5059, and the references cited therein) varieties of the sea hare Dolabella auricularia, from which a large number of antineoplastic and/or cytostatic linear and cyclic peptides, designated the dolastatins, have been isolated. Most of these potentially important peptides contain unprecedented amino acid units. Among these, the linear peptides, dolastatin 15 (1) (see: PETTIT et al., 1989a, Isolation and structure of the cytostatic linear depsipeptide dolastatin 15, Journal of Organic Chemistry, 54, 6005) and dolastatin 10 (2) (see: PETTIT et al., 1987, The isolation and structure of a remarkable marine animal antineoplastic constituent: Dolastatin 10, Journal of the American Chemical Society, 109, 6883) have exhibited the most potent antineoplastic activity (see: U.S. Pat. Nos. 4,816,444; 4,879,278; 4,978,744 and 5,554,725; and Hu et al., 1993, Effects of dolastatins on human B-lymphocytic leukemia cell lines, Leukemia Research, 17, 333) and have been selected for clinical development. Indeed, Phase 1 clinical trials of dolastatin 10 (2) have been ongoing under the auspices of the U.S. National Cancer Institute since November, 1995.
Since 1984, considerable research efforts have been directed to exploring structural modifications of dolastatin 10 (2) (PETTIT et al., 1995, Antineoplastic Agents 337, Synthesis of Dolastatin 10 Structural Modifications, Anticancer Drug Design, 10, 529) and dolastatin 15 (1) for the purpose of developing new potential anticancer drugs. A number of structural modifications of these peptides have been investigated in order to alter the antineoplastic activity of the parent molecule and eliminate from each peptide where possible, the more synthetically challenging units, especially the phenylalanine-derived C-terminal segments. Preliminary structure/activity studies based on dolastatin 10 (2) suggested that the thiazole-containing C-terminal unit could be adequately replaced with xcex2-phenethylamine without significant loss of activity, whereas certain other modifications led to moderate or more drastic loss of antiproliferative activity (see: PETTIT et al., 1995, Antineoplastic Agents 337, Synthesis of Dolastatin 10 Structural Modifications, Anticancer Drug Design, 10, 529) without significant change in inhibitory effects on tubulin assembly. In the case at hand, a series of structural modifications of dolastatin 15 were made in which the C-terminal dolapyrrolidinone unit (5) was replaced by various amides. In contrast to dolastatin 10, major structural changes in the C-terminal amide unit of dolastatin 15 had essentially no adverse affect upon the inhibitory effects of the depsipeptide against tumor cell growth, but did result in a moderate reduction in the inhibition of tubulin polymerization.
One major factor driving the present research arises from the inescapable fact that there is not enough Dolabella auricularia in the world to allow sufficient quantities of effective components to be isolated therefrom to meet the need of the cancer-afflicted population in a commercially feasible manner. Therefore, a commercially effective synthesis must be developed which is capable of replicating a molecule containing only those substituents which are effective to control or arrest or mitigate the spread of cancer cells through a human system inflicted therewith. It is toward that goal that the present invention is directed.
All amino acids (S-configurations) and derivatives which are discussed herein were used as obtained from Sigma-Aldrich Co. Other reagents (DEPC, DCC, EDC-HCl, HOBt, NMM, Et3N, 4-pyrrolidinopyridine, TFA, etc. {Abbreviations used: DEPC (diethylphosphorocyanidate), DCC (N,Nxe2x80x2-dicyclo-hexylcarbodiimide), EDC-HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), WRK (Woodward""s reagent K, 2-ethylphenylisoxazolium-3xe2x80x2-sulfonate), BroP (tris(dimethylamino) phosphonium bromide hexafluorophosphate), HOBt (1-hydroxybenzotriazole), NMM (4-methylmorpholine), Et3N (triethylamine), TFA (trifluoroacetic acid), THF (tetrahydrofuran), EtOAc (ethyl acetate), AcOH (acetic acid), Z (benzyloxycarbonyl), Boc (tert-butyloxycarbonyl)}) described were also obtained from Sigma-Aldrich and used without further purification. Amines 6a-r were either redistilled or recrystallized. All solvents were redistilled, and solvent extracts of aqueous solutions were dried over anhydrous magnesium sulfate or sodium sulfate. THF was distilled from LiAlH4. Reactions were monitored by thin-layer chromatography using ANALTECH silica gel GF (0.25 mm) plates visualized by either UV irradiation or 3% ceric sulfate in 3 N H2SO4 solution as appropriate. Crude products were purified by flash chromatography over silica gel (E. Merck, DARMSTADT, 70-230 mesh). The final peptide products (12a-r) were further purified by rapid gel permeation chromatography in methanol on a column of lipophilic SEPHADEX LH-20.
Melting points were measured with an ELECTROTHERMAL digital melting point apparatus, model IA9200, and are uncorrected. Optical rotation measurements were recorded on a PERKIN-ELMER 241 polarimeter in methanol (unless otherwise noted) at 25xc2x0 C. IR spectra were obtained using a NICOLET FTIR Model MX-1 instrument. All 1H-NMR spectra were observed on a VARIAN GEMINI 300 MHz instrument with CDCl3 or DMSO-d6 as solvent as noted. The 13C-NMR spectra were obtained with a UNITY 500 MHz instrument in CDCl3. EIMS data were recorded with a MAT 312 mass spectrometer. Elemental analyses were determined by Galbraith Laboratories, Inc. located in Knoxville, Tenn.
All compounds synthesized were first evaluated for in vitro antitumor activity against murine P388 lymphocytic leukemia cells and against murine L1210 leukemia cells and human CA46 Burkitt lymphoma cells using techniques described by Hamel and Lin (see: HAMEL, E., and LIN, C. M., 1993, Interaction of combretastatin, a new plant-derived antimitotic agent, with tubulin, Biochemical Pharmacology, 32, 3864). The compounds were also evaluated in the NCI""s human tumor 60-cell-line in intro primary screen as described by Monks et al. (see: MONKS, A., SCUDIERO, et al., 1991, New colorimetric cytotoxicity assay for anticancer drug screening, Journal of the National Cancer Institute, 83, 757). Data analyses were performed using methods described by Boyd and Paull (see: BOYD, M. R., et al., 1995, Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen, Drug Development Research, 34, 91). A tubulin polymerization inhibition assay was performed according to a modified version of the procedure used previously and described by Muzaffar et al. (see: MUZAFFAR, A., et al., 1990, Antitubulin effects of derivatives of 3-demethylthiocolchicine, methylthio esters of natural colchicinoids, and thioketones derived from thiocolchicine, Comparison with colchicine, Journal of Medicinal Chemistry, 33, 567) to evaluate the effects of dolastatin 15 on cellular microtubule assembly. The new assay for these compounds employed a drug-tubulin preincubation (15 min.) with 10 xcexcM tubulin in 0.8 M glutamate at 30xc2x0 C., followed by addition of GTP and incubation (20 min.) at 30xc2x0 C., with tubulin polymerization monitored turbidimetrically in GILFORD recording spectrophotometers.
Antimicrobial disk susceptibility tests were performed according to the method established by the National Committee for Clinical Laboratory Standards (NCCLS, 1997). MUELLER-HINTON agar was used for susceptibility testing of Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Escherichia coli, Enterobacter cloacae, Bacillus subtilis, Pseudomonas aeruginosa, and Erwinia carotovora, Gonococcal Typing agar for Neisseria gonorrhoeae, and YM agar for Candida albicans and Cryptococcus neoformans. Immediately prior to susceptibility assays, compounds were reconstituted in sterile DMSO and twofold dilutions applied to sterile 6-mm disks. Zones of inhibition were read at 16 hours for bacterial cultures (except for M. luteus) and 42 hours for fungal cultures and M. luteus. MIC determinations were performed in duplicate.